Cardiac pacemaker cells of the Sinoatrial Node are essential for producing rhythmic heartbeats. Despite the importance of this specialized cardiac cell type, little is known about their ontogeny or mechanisms of specification and differentiation. Our preliminary data show that although the primary heart tube initially displays rhythmic beating, the cells pacing it do not differentiate into the Sinoatrial Node. Instead, pacemaker precursors arise from the mesoderm posterior to the known cardiogenic field and take over the pacing function during heart-looping. Surprisingly, the pacemaker precursor mesoderm can differentiate in culture without any other surrounding embryonic tissues. They display sustained rhythmic beat rates, and sensitivity to pacemaker cell specific ion channel blockers. Furthermore, they have the ability to pace other cardiomyocyte populations. Our global RNA profiling has identified a unique set of Wnt-related genes expressed in pacemaker precursors. These somewhat surprising findings lead to three central hypotheses: (1) the pacemaker precursors arise from a specific mesoderm population separate from the heart field; (2) pacemaker cell fate is induced prior to heart tube formation; and (3) Wnt-related signaling, which is inhibitory to the heart field, acts as a promoting signal during pacemaker cell specification. We will test these hypotheses experimentally. This proposal will provide the first basis for identifying the embryonic origin of pacemaker precursors (Aim 1), the ability of a specific mesoderm subpopulations to enter the pacemaker cell fate (Aim 2), and novel molecular mechanisms that specify and direct the differentiation this important cell type during a brief temporal window and at a defined site in the embryo (Aim 3). PUBLIC HEALTH RELEVANCE: The cardiac pacemaker is essential for cardiac function and survival. Limited options of effective treatments contribute to the continued prevalence of arrhythmic heart disease. The proposed study will explore the molecular mechanisms that regulate the earliest developmental steps leading to the generation of this essential cardiac tissue and may provide a basis for future therapeutic approaches.